Inhibitors of kynureninase

ABSTRACT

The present invention provides inhibitors of kynureninase having the formula ##STR1## where X is CHOH, S, SO 2 , SO, SONH, PO 2  H or PONH 2 , R A  and R B , independently of one another, are H, a halogen, CF 3  or a small alkyl group having one to three carbon atoms; A is a H or an acetyl group; R 1  is H, NH 2 , NR 6  R 7 , NO 2 , halogen, CF 3  or a small alkyl group having from one to three carbon atoms, wherein: R 6  and R 7 , independently of one another, are H, a formyl group or a small alkyl group having from one to three carbon atoms with the exception that only one of R 6  or R 7  can be a formyl group; R 2  is OH, H, halogen, CF 3  or a small alkyl group having from one to three carbon atoms; and R 3 , R 4  and R 5 , independently of one another, are H, halogen, CF 3 , NO 2 , NH 2 , or small alkyl group having from one to three carbon atoms. In particular, compounds of this formula in which X is CHOH, S or SO 2  are provided. In compounds of this formula in which X is CHOH, those having the (αS,γS) configuration or the (αR,γR) configuration when R A  or R B  is a hydrogen, are more potent inhibitors of kynureninase. Inhibitors of mammalian kynureninase are of particular use in therapy for certain neurological disorders.

This invention was made through a grant from the National Institutes ofHealth. The United States Government has certain rights in thisinvention.

This application is a 371 PCT/US9203198, filed Apr. 17, 1992 acontinuation-in-part of U.S. Ser. No. 07/689,705, filed Apr. 18, 1991and issued as U.S. Pat. No. 5,254,725 on Oct. 19, 1993, and acontinuation-in-part of U.S. Ser. No. 07/840,408, filed Feb. 24, 1992now abandoned.

BACKGROUND OF THE INVENTION

Kynureninases are a group of pyridoxal-5'-phosphate dependent enzymeswhich catalyze the hydrolyric β,γ-cleavage of aryl-substitutedα-amino-γ-keto acids, particularly L-kynurenine or3-hydroxy-L-kynurenine to give L-alanine and anthranilic acid or3-hydroxyanthranilic acid, respectively (see: K. Soda and K. Tanizawa(1979)Advances Enzym. 49:1-40). Kynureninase is involved in themicrobial catabolism of L-tryptophan via the aromatic pathway. In plantsand animals, a kynureninase is required in tryptophan catabolism and forNAD biosynthesis via guinolinic acid. Quinolinic acid is a relativelytoxic metabolite which has been implicated in the etiology ofneurological disorders, including epilepsy and Huntington's chorea (R.Schwarcz et al. (1988) Proc. Natl. Acad. Sci. USA 85:4079; M. F. Beal etal. (1986) Nature 321:168-171; S. Mazzari et al. (1986) Brain Research380:309-316; H. Baran and R. Schwarcz (1990) J. Neurochem. 55:738-744).Inhibitors of kynureninase are thus important targets for treatment ofsuch neurological disorders.

L-kynurenine (which can also be designatedα,2-diamino-γ-oxobenzenebutanoic acid) is the preferred substrate ofbacterial kynureninase, which is exemplified by that of Pseudomonasfluorescens (O. Hayaishi and R. Y. Stanier (1952) J. Biol. Chem.195:735-740). The kynureninase of tryptophan metabolism in plants andanimals has a somewhat different substrate specificity with3-hydroxy-L-kynurenine (which can be designatedα,2-diamino-3-hydroxy-γ-oxobenzenebutanoic acid) being the preferredsubstrate (Soda and Tanizawa (1979) supra).

The mechanism of kynureninases has been the subject of considerableinterest due to the unique nature of this pyridoxal-5'-phosphatedependent reaction. Mechanisms based on redox reactions (J. B.Longsnecker and E. E. Snell (1955) J. Biol. Chem. 213:229-235) ortransamination (C. E. Dalgleish et al. (1951) Nature168:20-22) have beenproposed. More recently mechanisms involving either a nucleophilicmechanism with an "acyl-enzyme" intermediate (C. Walsh (1979) "EnzymaticReaction Mechanisms" W. H. Freeman and Co., San Francisco, p. 821; M.Akhtar et al. (1984) "The Chemistry of Enzyme Action" New ComprehensiveBiochemistry, Vol. 6 (M. I. Page, ed.) Elsevier, New York, p.821) or ageneral base-catalyzed mechanism (K. Tanizawa and K. Soda (1979) J.Biochem. (Tokyo) 86:1199-1209) have been proposed.

In addition to the physiological reaction, kynureninase has been shownto catalyze an aldol-type condensation of benzaldehyde with incipientL-alanine formed from L-kynurenine to giveα-amino-γ-hydroxy-γ-phenylbutanoic acid (G. S. Bild and J. C. Morris(1984) Arch. Biochem. Biophys. 235:41-47). The stereochemistry of theproduct at the γ-position was not determined, although the authorssuggested that only a single isomer was formed.

J. L. Stevens (1985) J. Biol. Chem 260:7945-7950 reports that rat liverkynureninase displays cysteine conjugate β-lyase activity. This enzymeactivity is associated with cleavage of s-cysteine conjugates of certainxenobiotics to give pyruvate, ammonia and a thiol, for example, cleavageof S-2-(benzothiazolyl)-L-cysteine to give 2-mercaptobenzothiazole,pyruvate and ammonia. More recently, I. S. Blagbrough et al. (1990)Toxicol. Lett 53(1-2):257-259 (Chem. Abstract 114(9):77537k) report thatcysteine conjugate β-lyase (C-S-lyase) is a member of a family oftransaminases and aminotransferases and that C-S lyase is a glutaminetransaminase K. The reference discusses structure-activity relationsdisplayed by C-S-lyases. C-S-lyases are distinguishable fromkynureninase but exhibit overlapping activities.

Several reports concerning the relative reactivities of kynurenineanalogs with bacterial kynureninase or rat liver kynureninase aresummarized in Soda and Tanizawa (1979) supra. Tanizawa and Soda (1979)supra reported that a number of ring substituted L-kynurenines, namely:3-hydroxy-, 5-hydroxy-, 5-methyl-, 4-fluoro-, and 5-fluoro-L-kynureninewere substrates of kynureninase of P. fluorescens. These authors alsoreported that dihydrokynurenine (called γ-(o-aminophenyl)-L-homoserinetherein) was a substrate for that kynureninase, yieldingo-aminobenzaldehyde and L-alanine. The K_(m) of dihydrokynurenine wasreported to be 67 μM compared to a K_(m) of 35 μM for L-kynurenine and200 μM for 3-hydroxy-L-kynurenine. N'-formyl-L-kynurenine andβ-benzoyl-L-alanine were likewise reported to be substrates (with K_(m)=2.2 mM and 0.16 mM, respectively) for the bacterial kynureninase.Tanizawa and Soda measured relative reactivity as relative amounts ofL-alanine formed.

O. Hayaishi ( 1955 ) in "A Symposium on Amino Acid Metabolism" (W. D.McElroy and H. B. Glass, eds.) Johns Hopkins Press, Baltimore pp.914-929 reported that 3-hydroxy- and 5-hydroxy-L-kynurenine,β-benzoyl-L-alanine and β-(o-hydroxybenzoyl)-L-alanine PG,5 weresubstrates for the bacterial enzyme, but that N'-formyl-L-kynurenine wasnot a substrate. O. Hayaishi measured relative reactivities bydetermining the amount of substrate hydrolyzed.

Tanizawa and Soda (1979) supra reported that S-benzoyl-L-cystsine,L-asparagine and D-kynurenine were not substrates of kynureninase, whileO. Hayaishi (1955) supra reported that β-(p-aminobenzoyl)-L-alanine,β-(o-nitrobenzoyl)-L-alanine, β-(m-hydroxybenzoyl)-L-alanine,3-methoxy-L-kynurenine, β-benzoylpropanoic acid, andβ-(o-aminobenzoyl)propanoic acid do not react with bacterialkynureninase. Kynureninase is reported to act only on L-amino acids (M.Moriguchi et al. (1973) Biochemistry 12:2969-2974).

O. Wiss and H. Fuchs (1950) Experientia 6:472 (see: Soda and Tanizawa(1979) supra) reported that 3-hydroxy-L-kynurenine, L-kynurenine,β-benzoyl-L-alanine, γ-phenyl-L-homoserine, γ-methyl-L-homoserine,2-aminolevulinic acid and α-amino-γ-hydroxypentanoic acid reacted withrat liver kynureninase to produce alanine, whileβ-(o-nitrobenzoyl)-L-alanins did not.

G. M. Kishore (1984) J. Biol. Chem. 259:10669-10674 has reported thatcertain β-substituted amino acids are mechanism-based inactivators ofbacterial kynureninase. Several β-substituted amino acids including:β-chloro-L-alanine, o-acetyl-L-serine, L-serine O-sulfate,S-(2-nitrophenyl)-L-cystsine (called S-(o-nitrophenyl)-L-cysteine,therein) and β-cyano-L-alanine inactivated kynureninase. Theseβ-substituted amino acids react with kynureninase to give pyruvate andammonia. However, a portion of the turnovers of the enzyme lead toformation of an inactive enzyme complex. S-(2-nitrophenyl)-L-cysteinewas described as the "most efficient suicide substrate at lowconcentrations" with a K_(i) of 0.1 mM.

Bacterial kynureninase is also strongly inhibited by o-aminobenzaldehyde(K_(i) =6.5 μM, non-competitive inhibition). Several other aromaticshaving "a carboxyl group on the benzene ring and an amino group at theortho-position" including o-aminoacetophenone, anthranilic acido-nitrobenzaldehyde and benzaldehyde were described as inhibitors(Tanizawa and Soda (1979) supra). It was suggested that inhibitionrelates to binding of the formyl group to the portion of the enzyme thatserves as a binding site for the γ-carboxyl of kynurenine. Anthranilateand 3-hydroxyanthranilate, the products of the kynureninase reaction,were also reported to inhibit the enzyme (Takeuchi et al. (1980) J.Biochem. (Tokyo) 88:987-994).

Blagbrough, I. S. et al. (1988) Drug Metab. Drug Interact 6(3-4):303-316in them. Abstracts 112(19):174617c report on inhibition of rat renal C-Slyase by certain cystsine conjugates. Certain S-(nitro-substitutedphenyl)-L-cysteines and N-acetyl-S-(nitro-substitutedphenyl)-L-cysteines were reported to inhibit C-S lyase as measured by akidney slice methodology. The nitrophenyl cystsine conjugates:S-(2-nitrophenyl)-L-cysteine, S-(4-nitrophenyl)-L-cysteine,S-(2,6-dinitrophenyl)-L-cysteine,N-acetyl-S-(3,4-dinitrophenyl)-L-cystsine,N-acetyl-S-(2,6-dinitrophenyl)-L-cysteine andN-acetyl-S-(2-chloro-4-nitrophenyl)-L-cysteine are reported to inhibitC-S layse.

Vamvakas et al. (1988) Chem. Biol. Interact 65:59-71 in Chem. Abstracts109(7):50020w refers to the cystsine conjugate β-lyase-mediatedmetabolism of certain cystsine conjugates including S-benzyl-L-cysteinewhich was reported to be cleaved to give pyruvate. The reference notesthat aminooxyacetic acid is an inhibitor of the β-lyase.

Tolosa et al. (1968) Mol. Biol. 2(5):769-777 [in Russian] in Chem.Abstracts 70(1):482d reported that cystsine lyase was significantlyinhibited by H₂ NOH and its O-substituted derivatives and thataminooxyacetic acid was the most inhibitory derivative tested.

J. P. Whitten et al. (1989) Tetrahedron Letts. 30:3649-652 reported thesynthesis of 2,2-difluoro-α-benzoyl alanine(α-amino-β,β-difluoro-γ-oxobenzene butanoic acid) which is said to be a"potential new inhibitor of kynureninase." Fluoroketone-containingpeptides are described as capable of forming stable hydrates orhemiketals which are "thought to inhibit" proteolytic enzymes as analogsof a tetrahedral transition state. The difluoro compound is described asa competitive inhibitor of kynureninase, but no details of thisinhibition are given in the reference.

The present work is based on a reexamination of the mechanism ofkynureninase catalysis, in particular, through an investigation of thestereospecificity of the retro-aldol reaction catalyzed by the enzyme.During the course of this work, the reactivity of dihydrokynurenine withkynureninase was found to be significantly different than had previouslybeen reported. The result of these mechanism and reactivity studies wasthe identification of a class of potent kynureninase inhibitors. Thepresent invention provides kynureninase inhibitors which are designed tobe "transition-state analogue" inhibitors.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide means andcompositions for inhibition of kynureninase. In the methods of thisinvention a kynureninase is contacted with an inhibitory amount of akynureninase inhibitor of this invention. The kynureninase inhibitors ofthis invention are amino acid derivatives of the formula: ##STR2##wherein the stereochemical configuration at the α-carbon is as indicatedin Formula I (and is the same configuration as the α-carbon inL-kynurenine ); where X is CHOH, S, SO₂, SO, SONH PO₂ H, or PONH₂ ;wherein R_(A) and R_(B), independently of one another are H, halogen,CF₃ or a small alkyl group having one to three carbon atoms; A is H oran acetyl group; R₁ is H, halogen, NH₂, NR₆ R₇, NO₂, CF₃, or a smallalkyl having from one to three carbon atoms; with R₆ and R₇,independently of one another, being H, a small alkyl group having fromone to three carbon atoms, or COH wherein only one of R₆ or R₇ can beCOH; R₂ is OH, H, halogen, CF₃ or a small alkyl having from one to threecarbon atoms; and R₃, R₄ and R₅, independently of one another, are H,OH, halogen, CF₃, NO₂, NH.sub. 2, or a small alkyl group having from oneto three carbon atoms, and with the proviso that the compound of formulaI is not S-(2-nitrophenyl)-L-cysteine.

A subset of inhibitors of this invention excludesS-(4-nitrophenyl)-L-cysteine, S-(2,4-dinitrophenyl)-L-cysteine,S-(3,4-dinitrophenyl)-L-cysteine, S-(2,6-dinitrophenyl)-L-cysteine,S-(2-chloro-4-nitrophenyl)-L-cysteine, or an N-acetyl derivativethereof.

For inhibition of kynureninase, X is preferably CHOH, S or SO₂ with CHOHand SO₂ being more preferred, and it is generally preferred that R₁ isNH₂.

Inhibitors useful in the methods of this invention include the compoundsof formula I in which the halogen of R₁ -R₅ is fluorine, R₂ is H or OH,R₁ is H or NH₂ ; and R_(A), R_(B), R₄ and R₅ are H or fluorine. Usefulinhibitors also include those in which R₃ is H, NH₂, NO₂ or fluorine,with H or fluorine preferred. More preferred inhibitors are those inwhich R₁ is NH₂ and R_(A), R_(B), R₃, R₄ and R₅ are H.

For inhibition of bacterial kynureninase, it is preferred that R₂ is H.For inhibition of plant and animal kynureninase, it is preferred that R₂is OH.

Subsets of inhibitors useful in the methods of this invention arecompounds of formula I in which:

X contains a S atom, including X=SO₂, SO, S, or SONH;

X contains a P atom, including X=PO₂ H or PONH₂ ;

X contains a C atom, including X=CHOH;

X is S and none of R₁ -R₅ is NO₂ ;

X is S and R₁ is NH₂, NR₆ R₇, halogen, CF₃ or a small alkyl group havingfrom one to three carbon atoms, and R₆ and R₇ are as defined above;

X is SO₂ and R₁ is NH₂, NR₆ R₇, halogen, CF₃ or a small alkyl grouphaving from one to three carbon atoms, and R₆ and R₇ are as definedabove;

X is SO and R₁ is NH₂, NR₆ R₇, halogen, CF₃ or a small alkyl grouphaving from one to three carbon atoms, and R₆ and R₇ are as definedabove;

A is H; or

X is SO₂, SO or CHOH and A is H or an acetyl group.

It is a specific object of the present invention to provide methods forinhibition of kynureninase which employ derivatives ofα-amino-γ-hydroxy-γ-hydroxybenzene butanoic acids of the formula:##STR3## wherein the stereochemical configuration at the α carbon is asindicated (and is the same configuration as the α-carbon inL-kynurenine), wherein R_(A) and R_(B), independently of one another areH, halogen, CF₃ or a small alkyl group having one to three carbon atoms;R₁ is H, halogen, NH₂, NR₆ R₇, NO₂, CF₃ or a small alkyl group havingfrom one to three carbon atoms, with R₆ and R₇, independently of oneanother, being H, CH₃ or COH, wherein only one of R₆ or R₇ can be COH;R₂ is OH, H, halogen, CF₃, or a small alkyl group having from one tothree carbon atoms; and R₃, R₄ and R₅, independently of one another, areH, OH, halogen, CF₃, NO₂, NH₂, or a small alkyl group having from one tothree carbon atoms. Inhibitors useful in the methods of this inventioninclude the compounds of formula II in which the halogen of R_(1-R) ₅ isfluorine, R₂ is H or OH, R₁ is NH₂ or H, and R_(A), R_(B), R₃, R₄ and R₅are H or fluorine. More preferred inhibitors are those in which R₁ isNH₂ and R_(A), R_(B), R₃, R₄ and R₅ are H.

For inhibition of bacterial kynureninase it is preferred that R₂ is H.For inhibition of plant and animal kynureninase it is preferred that R₂is OH.

It is a more specific object of this invention to provide methods ofinhibition of kynureninase which are α-amino-γ-hydroxy-γ-aryl butanoicacids having the structure: ##STR4## wherein the stereochemicalconfiguration at the α- and γ-carbons is as indicated (and theconfiguration at the α carbon is the same as that of the α-carbon inL-kynurenine) and wherein A, R₁₋₇, R_(A) and R_(B) are as defined abovefor formulas I and II. For inhibition of bacterial kynureninase it ispreferred that R₂ is H. For inhibition of plant and animal kynureninaseit is preferred that R₂ is OH.

It is a second specific object of this invention to provide methods ofinhibition of kynureninase employing an inhibitory amount of an S-arylderivative of L-cysteine which is an inhibitor of kynureninase and whichhas the formula: ##STR5## where the stereochemical configuration at theα-carbon is as indicated (and is the same as the α-carbon inL-kynurenine) where A, R₁₋₇, R_(A) and R_(B) are as defined for formulasI, II and III. Kishore (1984) supra had disclosed thatS-(2-nitrophenyl)-L-cysteine was a suicide inhibitor of kynureninase andBlagbrough et al. (1988) had disclosed that certain S-(nitro-substitutedphenyl)-L-cysteines and N-acetyl-S-(nitro-substitutedphenyl)-L-cysteines were inhibitors of cysteine conjugate β-lyase.

It is a further specific object of this invention to provide methods ofinhibiting kynureninase employing 3-arylsulfonyl-L-alanines (which canalso be designated S-aryl-L-cysteine sulfones) which are inhibitors ofkynureninase having the formula: ##STR6## where the stereochemicalconfiguration at the α-carbon is as indicated (the same as inL-kynurenine) and A, R₁.7, R_(A) and R_(B) are as defined for formulasI-IV.

Salts of the compounds of formulas I-V are considered functionalequivalents thereof with respect to inhibition of kynureninase. Inparticular, pharmaceutically acceptable salts of the compounds offormulas I-V are useful for the methods of the present invention and areuseful in any therapeutic treatment of animals based on the inhibitoryaction of the compounds of formulas I-V.

This invention thus provides methods of inhibiting kynureninase in vitroand/or in vivo which comprises the step of contacting the enzyme with aninhibitory amount of one or more of the compounds of formulas I-V orsalts, particularly pharmaceutically acceptable salts, thereof. It iswell understood in the art that a precursor prodrug may be converted invivo to a therapeutically active drug. Any such prodrug precursors ofthe compounds of formulas I-V are encompassed by this invention.

Therapeutic applications of the methods of the present invention relateparticularly to inhibition of animal kynureninases, particularly thoseof mammals. Inhibitors in which R₁ is NH₂ and R₂ is OH are preferred forsuch therapeutic applications.

Compounds of the present invention that are preferred for therapeuticapplications of the methods of the present invention are those that haveminimal toxic or irritant effect toward the target of the therapy. Ifthe inhibitor reacts with kynureninase, it is important that the productof that reaction be substantially nontoxic.

Kynureninases from different sources have different substratepreferences. For example, the preferred substrate of mammaliankynureninase is 3-hydroxy-L-kynurenine rather than L-kynurenine. Ingeneral, for a particular kynureninase, a preferred inhibitor of formulaI-V will possess the phenyl ring substitutions of a preferred substrateof that kynureninase. Substrate preferences of kynureninases are knownin the art or can be readily determined by routine experimentation.

Inhibitors of the present invention include, among others, ringfluorinated dihydrokynurenines: (αS,γS)- or(αS,γR)-α,2-diamino-γ-hydroxy-4-fluorobenzenebutanoic acid, (αS,γS)-- or(αS,γR)-α,2-diamino-γ-hydroxy-4-fluorobenzenebutanoic acid; ringhydroxylated dihydrokynurenines: (αS,γS)- or(αS,γR)-α,2-diamino-γ,5-dihydroxybenzenebutanoic acid; ring methylateddihydrokynurenines (αS,γS)- or(αS,γR)-α,2-diamino-γ-hydroxy-5-methylbenzenebutanoic acid, orring-substituted (αS,γS)- or (αS,γR)-α-amino-γ2-dihydroxybenzenebutanoicacid. Inhibitors of the present invention further includes N-acetylderivatives of the forgoing ring fluorinated dihydrokynurenines.

Inhibitors of kynureninase also include dihydrokynurenines:(αS,γS)-α,2-diamino-γ-hydroxybenzenebutanoic acid and(αS,γR)-α,2-diamino-γ-hydroxybenzenebutanoic acid;3-hydroxydihydro-kynurenines:(αS,γS)-α,2-diamino-γ,3-dihydroxybenzenebutanoic acid and(αS,γR)-α,2-diamino-γ,3-dihydroxylbenzenebutanoic acid anddihydrodesaminokynurenines: (αS,γS)-α-amino-γ-hydroxybenzenebutanoicacid and (αS,γR)-α-amino-γ-hydroxybenzenebutanoic acid.Dihydrokynurenine and dihydrodesaminokynurenine (see Soda and Tanizawa(1979) supra p. 32, Table VIII) were previously reported to besubstrates for certain kynureninases. Alternate substrates will act ascompetitive inhibitors toward the "natural" enzyme substrate.Dihydrokynurenine (Tanizawa and Soda (1979) supra) was reported to reactreadily with bacterial kynureninase with a reactivity about 65% that ofL-kynurenine. The dihydrokynurenine employed in that reference wasindicated to be a mixture of the (αS,γS) and (αS,γR) dihydrokynureninediastereomers. It was not disclosed therein and the data given thereindo not suggest that one of the diastereomers (αS,γS) is not a substratefor the kynureninase but acts as a competitive inhibitor of the enzymefor reaction of its natural substrates.

Inhibitors of the present invention further include N-acetyl derivativesof the forgoing dihydrokynurenines.

Subsets of inhibitors of this invention are compounds of formula IVwhich exclude one or more of the following combinations of phenyl ringsubstituents when R_(A) and R_(B) are both H:

R₁ -R₅ =H;

R₁ =NO₂ and R₂ -R₅ =H;

R₁ =NO₂, R₄ =Cl and R₂, R₃ and R₅ =H;

R₁ =NO₂, R₅ =NO₂ and R₂ -R₄ =H;

R₁ =NO₂, R₃ =CH₃, R₄ =CH₃ and R₂ and R₅ =H;

R₁ =NO₂, R₃ =CH₃ and R₂, R₄ and R₅ =H;

R₁ =NH₂ and R₂ -R₅ =H;

R₁ =Br and R₂ -R₅ =H;

R₁ =Br, R₃ =CF₃, and R₂, R₄ and R₅ =H;

R₁ =Br, R₂ and R₅ =OH and R₃ -R₄ =H;

R₁ =Cl, R₃ =Cl and R₂, R₄ and R₅ =H;

R₁ =Cl, R₃ =NO₂ and R₂, R₄ and R₅ =H;

R₁ =Cl, R₂, R₄ and R₅ =Cl, and R₃ =H;

R₁ -R₅ =Cl;

R₂ =NO₂, R₃ -NO₂ and R₁, R₄, and R₅ =H;

R₃ =F and R₁, R₂, R₄, and R₅ =H;

R₃ =NO₂ and R₁, R₂, R₄, and R₅ =H;

Other subsets of inhibitors of formula IV in which R_(A) and R_(B) are Hinclude those compounds in which:

R₁ =H, except when all of R₂ -R₅ =H, or when R₃ =halogen;

R₁ =NO₂, except when all of R₂ -R₅ =H, R₄ is a halogen, R₅ is NO₂, R₆ isNO₂, or R₃ is CH₃ ;

R₁ =NH₂, except when R₂ -R₅ =H;

R₁₌ halogen, except when R₂ -R₅ =H, R_(I) is OH or a halogen, R₃ is CF₃or R₄ is a halogen;

R₁ =NR₆ R₇ ;

R₁ =a small alkyl having from one to three carbon atoms; or

R₁ -R₆ ≠NO₂.

Subsets of inhibitors of this invention are compounds of formula V whichexclude one or more of the following combinations of phenyl substituentswhen R_(A) and R_(B) are both H:

R₁ -R₅ =H;

R₁ =CH₃, and R₂ -R₅ =H;

R₂ =CH₃, and R₁, R₃ -R₅ =H;

R₃ =CH₃, and R₁, R₂, R₄, R₅ =H;

R₃ =NO₂, and R₁, R₂, R₄, R₅ =H;

R₃ =NH₂, and R₁, R₂, R₄, R₅ =H; or

R₂ =CF₃, and R₁, R₃ -R₅ =H.

Another subset of inhibitors of formula V are those in which R_(A) andR_(B) are both H and in which R₁ -NH₂, NO₂, CF₃, halogen or NR₆ R₇ or inwhich R₁ -R₆ ≠NO₂.

A subset of inhibitors of formula V particularly useful for inhibitionof animal and plant kynureninases are those in which R₁ -NH₂, NO₂, CF₃,halogen or NR₆ R₇, and R₂ =OH. Compounds of formula V in which R₁ -NH₂and R₂ =OH are more preferred for inhibition of animal and plantkynureninases.

DETAILED DESCRIPTION OF THE INVENTION

Kynureninases catalyze the hydrolysis of aryl-substituted γ-keto-α-aminoacids. Kynureninase has been identified and isolated from certainbacteria, fungi, and yeasts as well as from mammalian sources.Kynureninases from different sources have been reported to havedifferent substrate specificities. L-kynurenine is the preferred"natural" substrate of bacterial kynureninase. In contrast, formammalian, yeast and fungal kynureninases, 3-hydroxy-L-kynurenine is thepreferred "natural" substrate. This preference for3-hydroxy-L-kynurenine, as assessed by relative substrate K_(m) 's, ischaracteristic of animal and plant kynureninase. The relative affinitiesof kynureninases for substrates other than L-kynurenine and3-hydroxy-L-kynurenine can also depend on the source of the enzyme.Animal and plant kynureninases are sometimes called3-hydroxykynureninases. The term kynureninase as used herein includesboth bacterial, plant and animal kynureninases. Bacterial kynureninasesare exemplified by the enzyme isolated from Pseudomonas fluorescens.Mammalian kynureninase is exemplified by the enzyme isolated frommammalian liver, in particular rat liver. A bacterial kynureninase willgenerally display substrate specificity like that of the P. fluorescenskynureninase. Mammalian kynureninase will generally display substratespecificity like that of rat liver kynureninase. Kynureninases, from allsources, catalyze the same types of reactions and so the mechanisms ofthe reactions they catalyze should be the same. Differences inaffinities for substrates is believed to be associated with differencesin the substrate binding site.

The present invention provides inhibitors of kynureninase. Some of theseinhibitors are substrates of the enzyme, some are not substrates. Manyof the inhibitors of this invention are competitive inhibitors of theenzyme for their natural substrates L-kynurenine and3-hydroxy-L-kynurenine.

Inhibition, as used herein, refers to inhibition of the hydrolysis ofL-kynurenine and/or 3-hydroxy-L-kynurenine. Competitive inhibition andnoncompetitive inhibition can be assessed by in vitro methods well-knownin the art. Preferred inhibitors of a particular kynureninase are thosehaving a K_(i) less than or equal to the K_(m) of the preferredsubstrate either L-kynurenine or 3-hydroxy-L-kynurenine for thatkynureninase. In general for competitive inhibitors, it is preferredthat the inhibitor have an affinity equal to or greater than that of thepreferred substrate for the enzyme. The level of inhibition that isachieved is dependent on the concentration of inhibitor in the vicinityof the enzyme. In general, the higher the affinity of the enzyme for theinhibitor, the more potent an inhibitor is. For applications of themethods of inhibition of kynureninase, particularly therapeuticapplications, it is generally preferred to employ high affinity (lowK_(i)) inhibitors to minimize the amount of inhibitor that must beadministered.

Kynureninases are known to catalyze other reactions, for example,cystsine conjugate β-lyase activity. Inhibition of kynureninases canalso be, at least qualitatively, assessed employing in vitro assays forsuch alternate kynureninase activities.

The aldol reaction of L-kynurenine and benzaldehyde catalyzed bykynureninase was found to proceed to give predominantly (80%) the (αS,γR) diastereomer of α-amino-γ-hydroxybenzenebutanoic acid.

The stereospecificity of the aldol reaction, as well as the results ofBild and Morris, Arch. Biochem. Biophys. (1984) 235:41-47, supports ageneral base mechanism for kynureninase, as shown in Scheme I. Thestereospecificity for cleavage of the (4R)-isomer is likely a reflectionof favorable orientation for the active site general base to initiatethe retro-aldol cleavage by proton abstraction (Scheme IA).

The basic group involved is probably the carboxylate that Kishore (1984)supra reported is modified by suicide substrate inhibitors. AlthoughKishore proposed that this carboxylate is responsible for α-protonabstraction, stereochemical studies by Palcic et al., J. Biol. Chem.(1985) 260:5248-5251, found that a α-proton of kynurenine is scrambledbetween the α- and β-positions of the L-alanine product, and thus theproton abstraction at the α-C is probably due to a polypprotic base,most likely a lysine ε-amino group. In the hydrolysis of L-kynurenine,the second general base would be required to assist in hydration of theketone, by abstraction of a proton from a water molecule (Scheme IB).The observed stereochemistry of the aldol-reactions suggests that thewater attacks on the reface of the carbonyl group, giving the(S)-gem-diolate anion. Subsequent rapid collapse of this tetrahedralintermediate is likely and would generate the enzyme-bound enamine ofPLP-L-alanine and anthranilic acid (Scheme IB). In the case of the(4S)-isomer, the carbinol group would mimic this gem-diol tetrahedralintermediate, but is not oriented in a position favorable for theretro-aldol reaction to occur. Thus, this compound is a"transition-state analogue," and would be expected to bind tokynureninase very tightly. ##STR7##

As an extension of these mechanistic studies, the reactivities ofdihydrokynurenine diastereomers were examined. (αS,γR)-Dihydrokynurenine((αS;γR)-α,2-diamino-γ-hydroxybenzenebutanoic acid) was found to be aslow substrate for the retro-aldol cleavage reaction catalyzed bykynureninase, while the analogous (αS,γS) diastereomer was unreactive.When these compounds were included in reaction mixtures of enzyme andL-kynurenine, the reaction was strongly inhibited. Analysis of thekinetic data in the presence of various concentrations of thedihydrokynurenines demonstrated that they act as competitive inhibitorswith respect to kynurenine, with K_(i) a values lower for the(αS,γS)-isomer 5 μM for the (αS,γR)-isomer. These can be compared to theK_(m) for L-kynurenine of 25 μM as measured in the present work, and thedata indicate that (αS,γS)-dihydrokynurenine binds about more tightlythan does L-kynurenine. This increased affinity of(αS,γS)-dihydrokynurenine is characteristic of mechanism-based, or"transition-state analogue" inhibitors.

The design of the kynureninase inhibitors of the present invention wasbased on the results of the inhibition studies on the diastereomers ofdihydrokynurenine in combination with what is known of substratespecificity of kynureninases.

Although not wishing to be bound by any specific theory, it is believedthat the inhibitors of the present invention represent "transition-stateanalogue" inhibitors of kynureninase in view of the newly proposedmechanism of Scheme I. Based on this proposed mechanismα-amino-γ-hydroxybenzenebutanoic acids having electron withdrawinggroups, including but not limited to, CF₃, halogen, NO₂, CN etc.appropriately substituted on the benzene ring to stabilize the proposed"transition state" will act as inhibitors of the kynureninase. Similary,S-aryl-L-cysteines and related compounds in which S is replaced by SO,SO₂, SONH, PO₂ H or PONH₂, which have electron withdrawing groupssubstituted on the aromatic ring, will stablize the proposed transitionstate and act as inhibitors of kynureninase.

The kynureninase inhibitors of the present invention can be prepared asexemplified for the preparation of the dihydrokynurenine diastereomersby selective reduction of the keto group of an appropriate γ-keto-aminoacid or by other methods well known in the art. Kynurenines, includingvarious ring-substituted kynurenines, can be prepared by ozonolysis oftryptophans. Alternatively, kynurenine analogs with desired ringsubstitution can be prepared enzymatically from appropriate tryptophansas described in Tanizawa and Soda (1979) supra and O. Hayaishi (1953) inBiochemical Preparations (E. E. Snell, ed.) Vol. 3, John Wiley & Sons,Inc., New York, pp. 108-111. The γ-keto amino acid,β-benzoyl-DL-alanine, can be prepared in several ways (for example, C.E. Dalgleish (1952) J. Chem. Soc. 137-141 and F. M. Veronese et al.(1969) Z. Naturforsch. 24:294-300) including amination ofβ-benzoylacrylate (Tanizawa and Soda (1979) supra). β-Benzoyl alanineshaving various desired ring substitution can be prepared using analogousmethods starting with appropriately substituted starting materials.Hayaishi (1955) supra and Wiss and Fuchs (1950) supra also providesources of γ -keto amino acids useful for preparation of the compoundsof the present invention. β-Benzoyl alanines can be selectively reducedby means known to the art to produce the inhibitors of the presentinvention.

Similarly, β-substituted γ-keto amino acids can serve as precursors tothe β-(or 2-)substituted γ-hydroxy amino acids of the present invention.Whitten et al. (1990) supra, provides a synthesis of2,2-difluoro-2-benzoyl alanine which can be selectively reduced to giveα-amino-β,β-difluoro-γ-hydroxybenzenebutanoic acid. Analogous methodscan be employed to prepare β-substituted, phenylring-substitutedγ-hydroxybenzenebutanoic acids of the present invention.

As will be appreciated by those in the art, reduction of a chiralnonracemic γ-keto amino acid, preferably an L-amino acid will generallyresult in a mixture of diastereomers. Techniques are available and wellknown in the art for the separation of diastereomers (HPLC, preparativeTLC, etc.).

S-(nitro-substituted phenyl)-L-cysteines (IV (NO₂)), were synthesized bynucleophilic aromatic substitution of fluoronitrobenzenes withL-cysteine in DMF in the presence of triethylamine (Phillips et al.(1989) Enzyme Microb. Techno. 11:80-83). The unsubstitutedS-phenyl-L-cysteine (IV (H)) was synthesized enzymatically following aprocedure by Soda et al. (1983) 47(12):2861 (Scheme II). This methodinvolves incubating thiophenol with L-serine in the presence oftryptophan synthase at 37.5° C. for 48 hrs. Reduction ofS-(nitrophenyl)-L-cysteines was accomplished by stirring with Zn dustand acetic acid.

The oxidation of thioethers to sulfones was achieved by using aprocedure described by Goodman et al. (1958) J. Org. Chem 23:1251, withslight modifications. This method produced good results when the arylcysteines were treated with a mixture of formic acid (98%) and hydrogenperoxide (30%). However, when 88% formic acid was used for this reactiona slightly impure product was obtained and the yields were also lower.The ease of formation of the sulfone depends on the position of nitrogroup on the ring. When a nitro group is present at the 2-position thecompletion of reaction took 48 hours or more, whereas, when there is nonitro group on the ring or when the nitro group is at the 4-position,the reaction is complete in 12 hours. Reduction of the nitro sulfoneswas performed by catalytic hydrogenation using acetic acid or formicacid as solvent (Scheme III).

Sulfoxide derivatives (I where X=SO) of the present invention can beprepared from known and readily available starting materials by meanswell-known to the art, for example, by oxidation of correspondingthioethers as described in Example 7 in the presence of a limitingamount of hydrogen peroxide.

Sulfoxamide derivatives of this invention can also be prepared fromknown and readily available starting materials by means well-known tothe art.

Phosphinate and phosphinamide derivatives (I where X=PO₂ H or PONH₂) canbe prepared from known and readily available starting materials by meanswell-known in the art, for example, by the Arbuzov reaction (Arbuzov.(1964) Pure Appl. Chem. 9:307-335) or routine modifications thereof.

N-acetyl derivatives of the present invention can be readily preparedfrom corresponding amines employing well-known techniques.

The results of competitive inhibition of certain thioether and sulfonecompounds are shown in Table 1. Among all the compounds tested, theunsubstituted S-phenyl-L-cysteine was found to be a very weak inhibitor,with K_(i) value of 0.7 mM, however, its oxidized analog,S-phenyl-L-cysteine sulfone, showed a 180-fold decrease in K_(i) valueto 3.9 μM. Similarly, substitution of an 2-amino group in theS-(2-aminophenyl)-L-cysteine showed a 318 fold decrease in K_(i) to 2.2μM. The compound which combined both of these structural features,S-(2-aminophenyl)-L-cysteine sulfone was found to be a considerably morepotent inhibitor of kynureninase, with K_(i) of about 70 nM. A similar,but less significant improvement in the activity of the compounds wereobserved by sulfone formation in the cases of 2-nitro,4-nitro and4-amino compounds. The results discussed above on the potent inhibitionby dihydrokynurenines indicate that the kynureninase reaction proceedsvia a gem-diol intermediate. The results of Table 1 indicate that theoxygens on the sulfur mimic the gem-diol tetrahedral transition state inthe reaction of L-kynurenine with kynureninase. Therefore, thesecompounds are examples of transition state analogs. The presence of anamine group and its position on the aromatic ring play an important rolein the activity of an inhibitor. When the amine group is moved from the2-position to the 4-position of the ring, the activity of the compounddrops 50-fold in case of cysteines and 120-fold in case of thecorresponding sulfones. This regiospecificity is expected, since the2-aminophenyl-L-cysteines are closer structural analogues of kynurenine.The presence of the nitro group on the ring decreases the activity ofall the compounds by several fold, possibly due to unfavorable stericinteractions.

Kishore (1984) supra disclosed that S-(2-nitrophenyl)-L-cysteineinactivates kynureninase. Although the author suggests on page 10674(second column) that "compounds similar to S-(o-nitrophenyl)-L-cysteineshould be ideal inactivators" and that "using an innocuous leaving groupwhich can provide specificity for interaction with the enzyme," noguidance or suggestion is found in the reference about what variationsin structure of S-(o-nitrophenyl)-L-cysteine can be made to give otherinhibitors. No specific guidance is given as to what constitutes an"innocuous" leaving group which retains "specificity for interaction."Blagbrough et al. (1988) supra discloses several nitro-substitutedS-phenyl L-cysteine and similarly substituted N-acetyl-L-cysteineinhibitors of cysteine β-lyase. Neither Kishore (1984) supra norBlagbrough et al. (1988) supra teach or suggest that oxidation of thesulfur of the cysteine in the disclosed compounds will result inkynureninase or cysteine β-lyase inhibition.

As has been described herein, one of the pair of diastereomers in cases,in which diastereomers can exist, will be a preferred kynureninaseinhibitor. It will be appreciated, however, that inhibition can beobtained by use of a mixture of the diastereomers. In order to obtainmaximal inhibition for the amount of inhibitor employed, it will bepreferable to maximize the amount of the more inhibitory diastereomer inthe mixture.

                  TABLE 1                                                         ______________________________________                                        Competitive Inhibition of Kynureninase.                                                          ##STR8##                                                   M                 Ki (μM)                                                  ______________________________________                                                ##STR9##      700                                                             ##STR10##     3.9                                                             ##STR11##     100                                                             ##STR12##     23                                                              ##STR13##     2.5                                                             ##STR14##     0.07.sup.1                                                      ##STR15##     --                                                              ##STR16##     12                                                              ##STR17##     140                                                     10.                                                                                   ##STR18##     8.5                                                     ______________________________________                                         .sup.1 This value is an upper limit K.sub.1 here is approximately the sam     order of magnitude as the concentration of enzyme in the assay, so that       the steadystate approximation may not apply.                             

EXAMPLES EXAMPLE 1 Investigation of the Mechanism ofKynureninase-catalyzed adol-reactions

Bacterial kynureninase was prepared from cells of Pseudomonasfluorescens (ATCC 11250, for example) essentially as described by.Hayaishi and Stanier (1952) J. Biol. Chem. 195:735-740. Cells weregrown on a minimal medium containing 0.1% L-tryptophan as the solecarbon and nitrogen source.

From 100 1 of medium, grown for 18 h at 30° C. 230 g of wet cell pastewas obtained. The cells were suspended in 1 of 0.01M potassiumphosphate,pH 7.0, and disrupted by 2 passages through a Manton-Gaulin homogenizer.After centrifugation of the cell extract for 1 h at 10000 g, the enzymewas partially purified by ion-exchange chromatography on DEAE-celluloseand ammonium sulfate precipitation. The preparation used in the resultsof Table 1 exhibited a specific activity of 0.2 μmol min⁻¹ mg⁻¹.

L-kynurenine and benzaldehyde (in excess) were incubated withkynureninase under the conditions described by Bild and Morris (1984)Arch. Biochem. Biophys. 235:41-47, which is incorporated by referenceherein. The product of this reaction was purified by preparative HPLCand identified as α-amino-γ-hydroxybenzenebutanoic acid. This productwas produced in quantitative yield based on L-kynurenine.

The α-amino-γ-hydroxybenzenebutanoic acid produced in the kynureninasereaction exhibited a negative CD (circular dichroism) extremum at 260nm, with vibronic splitting characteristic of a chirally substitutedbenzoyl alcohol chromophore. Based on a comparison of the CD spectra ofthe product with those of (R)- and (S)-mandelic acids, the predominantchiral product was determined to have the same absolute configuration as(S)-mandelic acid and thus to have the (γR)-configuration. (The terms Rand S are employed as is conventional according to theCahn-Ingold-Prelog rules.) NMR analysis (300 MH_(z) ¹ H) of the productdemonstrates that it is an 80:20 mixture of (αS,γR):(αS,γS)diastereomers of α-amino-γ-hydroxybenzene butanoic acid.

EXAMPLE 2 Reactivity of Dihydrokynurenine with Kynureninase

L-kynurenine (from commercial sources) was reduced with NaBH₄ in H₂ O togive dihydrokynurenine [α,2-diamino-γ-hydroxybenzenebutanoic acid]. Theprogress of reaction was monitored by the disappearance of the 360 nm UVabsorption band of L-kynurenine. The reduction resulted in a 60:40mixture of diastereomers. The diastereomers were separated bypreparative HPLC on a 20×250mm C18 column (Rainin, Dynamax) eluting with0.1% acetic acid (5 ml/min). The first peak to elute from the HPLCcolumn was identified by ¹ H NMR analysis to be the(αS,γS)-diastereomer. The second peak to elute was identified by ¹ H NMRanalysis to be the (αS,γR)-diastereomer.

The CD spectra of the separated dihydrokynurenine diastereomers wereconsistent with this identification.

The reactivity of the two dihydrokynurenines with kynureninase in 0.1Mpotassium phosphate buffer, pH 8.0, at 25° was examined. Reaction wasfollowed by the appearance of o-aminobenzaldehyde, as determinedspectrophotometrically by the increase in absorbance at 360 nm (SeeTanizawa and Soda (1979) Biochem. (Tokyo) 86:1199-1209, which isincorporated by reference herein).

The (αS,γR)-dihydrokynurenine diastereomer reacted slowly withkynureninase to produce o-aminobenzaldehyde. No significant reaction ofthe (αS,γS)-diastereomer was detected. Tanizawa and Soda (1979) suprahad reported that dihydrokynurenine reacted with kynureninase with aV_(max) of about 65% that of L-kynurenine. In contrast, the present workindicates that only the (αS,γR)-diastereomer of dihydrokynureninereacts, only at about 5% of the rate of L-kynurenine. Under theconditions employed and with the bacterial kynureninase prepared asdescribed in Example 1, K_(m) of the reaction of L-kynurenine wasdetermined to be 25 μM. This value is similar to the K_(m) of 35 μM forL-kynurenine obtained by Tanizawa and Soda.

EXAMPLE 3 Inhibition Kynureninase by Dihydrokynurenine

Inhibition of kynureninase by dihydrokynurenine was measured byincluding the potential inhibitor in the enzyme assay mixture (seeExample 1 and Tanzawa and Soda (1979) supra) and determining theapparent Km for L-kynurenine (the preferred substrate of bacterialkynurenine) in the absence and presence of the potential inhibitor.K_(i) values were then calculated using the standard equation:

    (K.sub.m).sub.app =K.sub.m (1+[I]/K.sub.i)

where [I] is the molar concentration of inhibitor and K_(m) =25 μM.

Inhibition of kynureninase by the (αS,γR)- and (αS,γS)-diastereomers ofdihydrokynurenine was examined and K_(i) 's were determined. Bothcompounds strongly inhibited the reaction of kynureninase withL-kynurenine. The K_(i) value for the (αS,γR)-diastereomer was lowerthan for the (αS,γR)-diastereomer. Both compounds were found to becompetitive inhibitors of kynureninase.

Inhibition of mammalian kynureninase can be measured using severaldifferent assays for enzyme activity. Rat liver kynureninase is obtainedfrom homogenization of rat liver, followed by precipitation with (NH₄)₂SO₄, as described by Stevens, J. L., J. Biol. Chem. (1985)260:7945-7950, which is incorporated by reference herein. The activityof rat liver kynureninase was assessed by measurement of the cysteineconjugate β-lyase activity, as described by Stevens (supra), withS-(2-benzothiazolyl)cysteine, a nonphysiological chromophoric substrate.Inhibition of kynureninase by the dihydrokynurenine diastereomers wasassessed with respect to reaction with that substrate.

Both the (αS,γR) and (αS,δS) diastereomers of dihydrokynurenine werefound to inhibit the reaction of rat liver kynureninase. The (αS,γS)diastereomer was found to be the stronger competitive inhibitor withK_(i) under the assay conditions of about 690 μM.

EXAMPLE 4 Synthesis of S-(phenyl)-L-cysteines (IV(H))

A mixture containing 1.23 ml (12 mM) of thiophenol, 0.525 g (5 mM) ofL-serine, 10 μM of potassium phosphate buffer, pH 7.8, 0.13 mg (20 nM)of pyridoxal-5'-phosphate and 5 mg of tryptophan synthase in a totalvolume of 25 ml was stirred at 37.5° C. After 48 hours the reactionmixture was cooled, the thick white precipitate was filtered and washedwith water and ethanol to give 0.31 g of white crystals ofS-(phenyl)-L-cysteine.

Tryptophan synthase was purified from cells of E. coli CB149 withplasmid pSTB7 containing the trpA and trpB genes from Salmonellatyphimurium, as described by Miles et al. (1989) J. Biol. Chem.264:6280.

EXAMPLE 5 Synthesis of S-(nitrophenyl)-L-cysteines (IV(NO₂))

To a flask containing 5 g of L-cysteine, 4.47 g of fluoronitrobenzeneand 20 ml of DMF was added 7.84 ml of triethylamine. After stirring atroom temperature for 3-4 hours, the contents of the flask solidifed intoa thick yellow cake. This solid was mixed with 15-20 ml of water andfiltered to give crude S-nitrophenyl-L-cysteine. Recrystalization fromhot water gave lemon yellow crystals of the product.

EXAMPLE 6 Synthesis S-(aminophenyl)-L-cysteines (IV(NH₂))

0.4 g of the S-nitrophenyl compound was dissolved in 50 mL of aceticacid, 2.0 g of zinc dust was added, and the mixture stirred at roomtemperature overnight. After completion of the reaction, the solid wasfiltered on celite and the filtrate was concentrated in vacuo to give anoil. This oil was triturated with water and methanol to give an offwhite solid of the reduced compound.

EXAMPLE 7 Synthesis of S-phenyl and S-nitrophenyl-L-cysteine sulfones(V(H) and V(NO₂))

0.65 g of S-phenyl or S-nitrophenyl compound was dissolved in 20 ml of98% formic acid and 4 ml of 30% hydrogen peroxide, and the mixturestirred at room temperature for 12-48 hours, depending on the compoundas discussed above. After completion of the reaction, the solvent wascarefully evaporated in vacuo at 25°-30° C. to give a white solid of thedesired product.

EXAMPLE 8 Synthesis of S-(aminophenyl)-L-cysteine sulfones (V(NH₂))

0.4 g of nitrophenyl sulfone was dissolved in 50 ml of formic acid,0.045 g of 10% Pd-C was added, and the mixture hydrogenated for 30minutes. The charcoal was removed by filtration on celite and thefiltrate was concentrated in vacuo to give a light tan oil, which upontrituration with methanol gave a light tan solid of the aminophenylsulfone.

EXAMPLE 9 Competitive Inhibition of Kynureninase by Compounds (IV and V)

Kynureninase activity was measured at 25° C. by following the decreasein absorbance at 360 nm (ε=-4500M⁻¹ cm⁻¹). A typical assay mixturecontained 0.4 mM L-kynurenine in 0.04M potassium phosphate, pH 7.8,containing 40 μM pyridoxal-5'-phosphate, at 25° C. The reactions ofS-aryl cysteines and S-aryl cysteine sulfones with kynureninase wereperformed using a spectrophotometric coupled assay with lactatedehydrogenase and NADH, by monitoring a decrease in absorbance due topyruvate formation. A typical assay mixture contained 30 μl lactatedehydrogenase solution (2 mg/ml), 0.1 mM NADH, 40 μMpyridoxal-5'-phosphate, 0.04M tris.HCl buffer, pH 7.8, with varyingconcentrations of the compounds. The competitive inhibition of thesecompounds was measured by variation of L-kynurenine concentrations atseveral fixed values of the inhibitor. K_(m) and V_(max) values werecalculated by fitting of initial rate data to the Michaelis-Mentenequation with ENZFITTER (Elsevier) on a Z-286 personal computer. KIvalues were determined from the equation:

    v=V.sub.max[S]/ (K.sub.m (1+[I]/K.sub.i)+[S]

Results for certain compounds of formulas IV-IX are given in Table 1.

Those of ordinary skill in the art will understand that alternative orequivalent methods, procedures, techniques and assays other than thosespecifically described herein can be readily employed or adapted toachieve the objects of this invention. All such alternative andequivalents are encompased by this invention. The scope of thisinvention is not limited by the specific examples herein which areintended to illustrate the invention.

We claim:
 1. A method for inhibiting kynureninase which comprises thestep of contacting said kynureninase with an inhibitory amount of acompound selected from the group consisting of compounds having theformula: ##STR19## and pharmaceutically acceptable salts thereof,wherein: X is S, SO₂, SO, SONH, PO₂ H or PONH₂ ;R_(A) and R_(B),independently of one another are H, a halogen, CF₃ or a small alkylgroup having one to three carbon atoms; A is H or an acetyl group; R₁ isH, NH₂, NR₆ R₇, NO₂, halogen, CF₃ or a small alkyl group having from oneto three carbon atoms, wherein:R₆ and R₇, independently of one another,are H a formyl group or a small alkyl group having from one to threecarbon atoms with the exception that only one of R₆ or R₇ can be aformyl group; R₂ is OH, H, halogen, CF₃ or a small alkyl group havingfrom one to three carbon atoms; and R₃, R₄ and R₅, independently of oneanother, are H, OH, halogen, CF₃, NO₂, NH₂ or small alkyl group havingfrom one to three carbon atoms, with the exception that the compound ofthe given formula is not S-(2-nitrophenyl)-L-cysteine.
 2. The method ofclaims 1 wherein said inhibitor is not S-(4-nitrophenyl)-L-cysteine,S-(2,4-dinitrophenyl)-L-cysteine, S-(3,4-dinitrophenyl)-L-cysteine,S-(2,6-dinitrophenyl)-L-cysteine, S-(2-chloro-4-nitrophenyl)-L-cysteine,or an N-acetyl derivative thereof.
 3. The method of claim 1 wherein X isSO₂, SO, SONH, PO₂ H or PONH₂.
 4. The method of claim 1 wherein X isSO₂.
 5. The method of claim 1 wherein X is S or SO₂.
 6. The method ofclaim 1 wherein:R_(A) and R_(B), independently of one another are H orF; R₁ is NH₂, H or F; R₂ is OH, H, or F; and R₃, R₄ and R₅,independently of one another, are H or F.
 7. The method of claim 6wherein R₁ is NH₂.
 8. The method of claim 7 wherein R₂ is H.
 9. Themethod of claim 7 wherein R₂ is OH.
 10. The method of claim 6wherein:R_(A), R_(B), R₃, R₄ and R₅ are --H, R₁ is NH₂ or H; and R₂ isOH or H.
 11. The method of claim 10 wherein R₁ is NH₂.
 12. The method ofclaim 11 wherein R₂ is H.
 13. The method of claim 11 wherein R₂ is OH.14. The method of claim 1 wherein in said inhibitory compound X≠S. 15.The method of claim 1 wherein said inhibitory compound is not aS-(nitro-substituted phenyl)-L-cysteine or N-acetyl derivative thereof.16. The method of claim 1 wherein said kynureninase is a mammaliankynureninase and in said compound R₁ is NH₂ and R₂ is OH.
 17. The methodof claim 1 wherein A is H.
 18. The method of claim 1 wherein X is SO₂.19. The method of claim 1 wherein X is S.
 20. A kynureninase inhibitorselected from the group consisting of compounds having the formula:##STR20## wherein X is S, PO₂ H or PONH₂ ; R_(A) and R_(B),independently of one another are H, a halogen, CF₃ or a small alkylgroup having one to three carbon atoms;A is H or an acetyl group; R₁ isH, NH₂, NR₆ R₇, NO₂, halogen, CF₃ or a small alkyl group having from oneto three carbon atoms, wherein:R₆ and R₇, independently of one another,are H, a formyl group or a small alkyl group having from one to threecarbon atoms with the exception that only one of R₆ or R₇ can be aformyl group; R₂ is OH, H, halogen, CF₃ or a small alkyl group havingfrom one to three carbon atoms; and R₃, R₄ and R₅, independently of oneanother, are H, halogen, CF₃, NO₂, NH₂ or small alkyl group having fromone to three carbon atoms with the exception that the compound of thegiven formula is not S-(2-nitrophenyl)-L-cysteine,S-(4-nitrophenyl)-L-cysteine, S-(2,4-dinitrophenyl)-L-cysteine,S-(3,4-dinitrophenyl)-L-cysteine, S-(2,6-dinitrophenyl)-L-cysteine,S-(2-chloro-4-nitrophenyl)-L-cysteine, or an N-acetyl derivative thereofand when X=S, R₁ -R₅ cannot each be H or a halogen; when X=S, R₁ cannotbe CH₃ or NH₂ when R₂ -R₅ are each H; when X=S, R₂ cannot be CH₃ when R₁and R₃ -R₅ are each H; when X=S, R₃ cannot be a halogen, CH₃, NH₂ or NO₂when X=S, R₁ -R₂ and R₄ -R₅ are each H.
 21. The inhibitor of claim 20wherein:R_(A) and R_(B), independently of one another are H or F; R₁ i sNH₂, H or F; R₂ is OH, H, or F; and R₃, R₄ and R₅, independently of oneanother, are H or F.
 22. The inhibitor of claim 21 wherein R₁ is NH₂.23. The inhibitor of claim 22 wherein R₂ is H.
 24. The inhibitor ofclaim 22 wherein R₂ is OH.
 25. The inhibitor of claim 21 wherein:R_(A),R_(B), R₃, R₄ and R₅ are H; R₁ is NH₂ or H; and R₂ is OH or H.
 26. Theinhibitor of claim 25 wherein R₁ is NH₂.
 27. The inhibitor of claim 26wherein R₂ is H.
 28. The inhibitor of claim 26 wherein R₂ is OH.
 29. Theinhibitor of claim 20 in which R₃ is NO₂.
 30. The inhibitor of claim 25in which R₁ is NH₂ and R₂₋₅, R_(A) and R_(B) are all H.
 31. Theinhibitor of claim 25 in which R₁₋₅, R_(A) and R_(B) are all H.
 32. Theinhibitor of claim 20 in which X is S and R_(A) and R_(B) are both H andwhen R₁ =H, all of R₂ -R₅ ≠H and R₃ ≠halogen or NO₂ ; when R₁ =NO₂, allof R₂ -R₅ ≠H, R₃ ≠CH₃, R₄ ≠halogen, R₃ ≠NO₂, and R₅ ≠NO₂ ; when R₁=halogen, all of R₂ -R₅ ≠H, R₃ ≠CF₃ or NO₂, R₄ ≠halogen and R₅ ≠OH or ahalogen; or when R₂ =NO₂, R₄ ≠NO₂.
 33. The inhibitor of claim 20 inwhich X is S, R₁ is NH₂ and R₂ is OH.
 34. The inhibitor of claim 20 inwhich A is H.
 35. The inhibitor of claim 20 which is not aS-(nitro-substituted phenyl)-L-cysteine.